The long term objective of this work is to develop a molecular understanding of the process by which centrosomes organize microtubules. The spatial orientation of microtubules is crucial for their function and is controlled by the centrosome. The centrosome has been recalcitrant to investigation so the basis for its function and regulation are unknown. We have discovered a highly conserved centrosome protein called pericentrin that is essential for cell division and organization of the microtubule cytoskeleton. Pericentrin provides us with a unique opportunity to understand centrosome function at the molecular level. A recent advance in our laboratory suggests that we will be able to identify and reconstitute the components of nucleation. We are now in an excellent position to: 1. Test the hypothesis that pericentrin is involved in centrosome assembly and define the sequence requirements for function. 2. Test the hypothesis that pericentrin assembles onto centrosomes in a cell cycle-specific phosphorylation-dependent manner. 3. Test the hypothesis that pericentrin binding proteins are involved in microtuble nucleation. Significant progress has been made toward th goals of this proposal. We have raised antibodies that block pericentrin function, produced mutant and wild type pericentrin cDNAs and fusion proteins and developed a novel in vitro assay for reconstituting centrosome function. Since that first submission of this proposal, we identified a pericentrin binding partner, perturbed centrosome structure using mutant pericentrin, and made the remarkable observation that pericentrin antibody-beads promote assembly of microtubules. These recent advances, molecular tools, and assays will allow us to make rapid progress in understanding the properties and function of pericentrin and centrosomes in general. Understanding centrosome function is important for several reasons. As the organizer of the microtubule cytoskeleton, the centrosome is a pivotal player in many fundamental cellular processes. Given its importance in the assembly of the mitotic spindle during cell division, the centrosome is a potential regulator of cell growth and a coordinator of morphogenesis. Centrosome- mediated microtubule polarity is critical for organizing the cytoplasm for intracellular vesicular transport and positioning of organelles. Despite its central position at the focus of microtubules and its central role in the life of a cell, the centrosome is poorly understood. Our preliminary results suggest that pericentrin will allow us to make important contributions in an area of biology that has been difficult to study. We are especially excited about the future prospect of realizing our long term goal of reconstituting centrosome function in vitro from individual components.